This grant proposes experiments to resolve critical viral DNA packaging initiation and translocation issues. Bacteriophage T4 DNA packaging mechanisms are widely shared among comparable phages packed with dsDNA through a prohead portal by comparable motor proteins (2), where the large terminase subunit (TerL) is responsible for translocation and the small terminase (TerS) for packaging initiation-cutting of the concatemer. Specific Aim 1 will establish the large gp17 (TerL) translocation mechanism. We propose a DNA crunching linear motor mechanism that employs a grip-and-release transient spring-like compression of B- to A-form- DNA. Our FRET measurements directly support this mechanism in a packaging stalled Y-DNA substrate in vitro that show a decrease in distance from terminase to portal; furthermore, there is a decrease in distance between closely positioned dye pairs in the Y-stem DNA that conforms to B- and A- structure. In normal translocation the TerL motor expels all B-form tightly binding YOYO-1 dye that cannot bind A-form. The motor cannot package A-form dsRNA or A-form DNA:RNA heteroduplexes. Our work shows that addition of helper B- form DNA:DNA (D:D) 20mers allows (D:R) packaging of heteroduplex A-form DNA:RNA 20mers (D:R), additional evidence for a B- to A-form spring motor. Additionally, kinetic analyses of fluorescent dye release, TerL cross-linking of photo-linkable dye, and high resolution structural data will provide support and insight into this proposed B-form to A-form motor mechanism. Crystallography and cryo-EM of TerL domains docked to proheads, portals, and to a clip region of the portal will confirm that the C-terminal nuclease domain of the terminase docks to the portal, as shown by FRET and SDM analysis. Specific Aim 2 will establish the role of the small terminase subunit gp16 (TerS) of phage T4 in DNA pac site interaction and in packaging initiation by a twin TerS ring mechanism. FRET measurements and superresolution microscopy will confirm that the T4 TerS protein acts in a double ring form to initiate packaging. Functional TerS-GFP and TerS-mCherry fusion proteins in vitro and in vivo serve as standards. FRET work shows that a ts mutant form of the TerS protein forms rings at low but not high temperature, showing ring formation is required for function. How do the double 22mer and single 11mer rings found in TerS protein-only preparations relate to DNA packaging? Strong genetic evidence supports synapsis of two homologous pac DNAs by a twin ring form of the TerS that opposes a four stranded pac DNA structure to judge by Holliday junction strand swapping DNA concatemer maturation for packaging initiation.